The sequencing of proteins from the carboxyl terminus has been a challenging problem in protein structure determination. Many methods for N-terminal sequencing have been published and readily available to the skilled artisan but little has been accomplished relative to the C-terminus, and fewer methods have resulted in commercial usage.
The thiocyanate method, described by Schlack et al., Physiol. Chem., 154: 125-170 (1926) involved the reaction of a protein or peptide with certain isothiocyanate reagents, in the presence of acetic anhydride, to form a C-terminal thiohydantoin amino acid. The derivatized amino acid is hydrolyzed to yield a shortened polypeptide and a thiohydantoin amino acid. The thiohydantoin amino acids are now analyzed by HPLC methods. A disadvantage of this reaction is the severity of the conditions required for complete derivatization of the C-terminal amino acid.
Stark, G. R., Biochemistry, 7: 1796-1807 (1968) introduced use of the reagent acetohydroxamate as one more mild and capable of performing a more rapid cleavage. U.S. Pat. No. 4,837,165 discloses the use of the reagent trimethylsilylisothiocyanate (TMS-ITC) which resulted in an improved yield of the thiohydantoin formation and reduced the number of side products obtained. The yields were low upon repetition which limited the number of degradation cycles to be performed. Further, not all amino acids were able to form thiohydantoin derivatives by this method.
Bailey et al., U.S. Pat. No. 5,180,807; discloses the use as a sequencing agent of an isothiocyanate regent which is a combination of phosphoroisothiocyanatidate and pyridine. The phophoroisothiocyanatidate includes a diphenyl moiety of the formula (PhO).sub.2 --P(O)--NCS or a diethyl derivative (EtO).sub.2 --P(O)--NCS. Bailey et al, U.S. Pat. No. 5,227,309 discloses using an alkyl or aryl tin isothiocyanate derivative, such as R.sub.x Sn(NCS).sub.y. Barley et al., U.S. Pat. No. 5,254,475 discloses use of alkali metal salts of trialkyl silanols and trialkylamine N-oxides, utilized in preference with silyl isothiocyanate as the coupling reagent.
Boyd et al., U.S. Pat. No. 5,185,266 discloses a method for cleaving the acyl thiohydantoin bond with an alkylating agent to form an adduct on the thiohydantoin. The adduct containing acyl-thiohydantoin is cleaved by reaction under substantially anhydrous acidic conditions.
Boyd et al., U.S. Pat. No. 5,051,368 discloses a method for forming thiohydantoins by activation of the amino acid with a ketenimine, and converting the ester to a thiohydantoin by reaction with a silyl or pyridine isothiocyanate.
Boyd et al., U.S. Pat. No. 5,041,388 discloses use of a mixed anhydride of isothiocyanic acid and a carboxylic or carbonic acid, for use under basic condition which reacts the peptide with the activated support derivatized as noted.
Boyd et al., U.S. Pat. No. 5,304,497 discloses a method of forming an N-protected amino acid for use in C-terminal peptide sequencing which utilizes uronium compounds with preferred thiocyanate, such as TMS-ITC, or crown ether adducts of metallothiocyanates.
Miller, U.S. Pat. No. 4,935,494 discloses phophoryl (thio)amide coupling reagents to yield arylthiohydantoin derivatives of amino acids. Miller et al., U.S. Pat. No. 5,066,785 discloses a coupling reagent for use in C-terminal peptides of the formula (R.sub.1).sub.n X.sub.a --P(.dbd.X.sub.c)((R.sub.2).sub.n X.sub.b)N(R.sub.3)--C(.dbd.X.sub.d)--X.sub.e.
Patent Applications to Hawke et al., U.S. Pat. No. 5,049,507 discloses a method of C-terminal sequencing wherein the peptide is reacted with a mixed anhydride of isothiocyanic acid and a carboxylic, carbonic or sulfonic acid under basic conditions.
Various substrate materials for use in sequencing C-terminal peptides have been proposed by Bailey et al., in U.S. Pat. No. 5,306,781 which discloses an activated carboxylic acid modified polyethylene membrane and in Sherrington et al., in U.S. Pat. No. 5,066,784 which discloses a porous polymeric material.